Quantification of supplemental enzymes in animal feedingstuffs by radial enzyme diffusion

Methods are described which facilitate quantification of supplemental cellulase, protease and alpha-amylase when added to animal feedingstuffs at normal industrial inclusion levels. The methods entail extraction of the enzymes from the feedingstuffs by agitation in buffer followed by quantification of extract activity using radial diffusion techniques. A linear relationship between the diameter of the zone of hydrolyzed substrate and the log of the enzyme activity applied is observed over a broad activity range. Assay of a feedingstuff supplemented with 1 kg t(-1) cellulase, protease and alpha-amylase yielded net supplemental activity recoveries of 104+/-11.7%, 91.3+/-6.74% and 126+/-29.5%, respectively. A similar assay method did not prove sufficiently sensitive to facilitate detection of xylanase at typical in-feed inclusion levels. The levels of endogenous cellulase, protease and alpha-amylase activity detected in the unsupplemented feedingstuffs were equivalent to 6.4+/-0.47%, 6.6+/-0.82% and 29.0+/-14.1%, respectively, of a 1 kg t(-1) supplement. The methods are technically straightforward and will facilitate determination of enzyme stabilities during processes such as high-temperature pelleting of feedingstuffs, as well as allowing more rigorous quality control related to enzyme-supplemented animal feedingstuffs.